The purpose of this research is to understand the mechanisms responsible for bile formation and the pathogenesis of disorders that result in impaired bile flow. The principal determinants of canalicular bile flow are bile acids and glutathione, which are secreted across the canalicular membrane by the bile salt export pump BSEP and the organic anion transporter MRP2, respectively. Secretion in many types of cells is regulated by cytoplasmic Ca2+, but a role for Ca2+ signaling in the regulation of BSEP or MRP2 has not been established. In the hepatocyte, Ca2+ signals are mediated entirely by inositol trisphosphate (InsP3), which acts through binding to the InsP3 receptor (InsP3R). The type II InsP3R (InsP3R-2) is the predominant isoform in hepatocytes, and it is concentrated near the canalicular membrane. However, there is also evidence that canalicular secretion is mediated by cAMP. Because adenylyl cyclase 6 (AC6) associates with InsP3R-2, this suggests that bile secretion mediated by Ca2+ and cAMP may be linked. The hypothesis of this proposal is that canalicular bile formation is Ca2+-dependent, in that it relies on local activation of peri-canalicular type II InsP3Rs by cAMP produced by AC6, which results in Ca2+-mediated insertion of MRP2 and BSEP into the canalicular membrane. It is further hypothesized that loss of these peri-canalicular InsP3Rs will impair regulated delivery of these transporters to the canalicular membrane, and thus lead to cholestasis. This hypothesis will be tested through three specific aims: (1) The role of impaired Ca2+ signaling in the pathogenesis of canalicular cholestasis will be determined. Expression and localization of InsP3R-2, MRP2, and BSEP, plus bile flow and composition will be investigated in animal models of canalicular cholestasis. The effects of impaired Ca2+ signaling or knockdown of InsP3R-2 or AC6 on secretion of organic anions and bile acids will be monitored directly in isolated hepatocytes and in perfused liver preparations; (2) How Ca2+ signals enhance canalicular targeting and membrane insertion of MRP2 and BSEP will be determined. Regulation of targeting and insertion of native transporters will be assessed in hepatocytes using immunoblot and biotinylation, and targeting and insertion of fluorescently tagged transporters will be assessed in liver cell lines using confocal, two-photon, and TIRF microscopy; and (3) The mechanism by which cAMP enhances Ca2+ signals in hepatocytes will be determined. We will use immunoprecipitation, FRET, and confocal fluorescence microscopy to determine whether AC6 and InsP3R-2 associate, and we will use RNA interference and Ca2+ imaging to determine whether and how this association enhances Ca2+ signaling; Together, these studies should identify the molecular and cellular basis for Ca2+ signaling in hepatocytes, and define how these signaling mechanisms in turn regulate bile secretion. This work should lead to an improved understanding of the mechanisms responsible for cholestasis, and should more generally provide a paradigm for the way in which Ca2+ signals are generated within specific microdomains in order to regulate epithelial secretion.